Apoptosis has been proposed as a mechanism for the T-cell depletion seen in individuals infected with HIV J. C. Ameisen and A. Capron, Immunol. Today, 12:102-105 (1991)!, as well as individuals having other pathological conditions. Apoptosis is accompanied by characteristic morphologic changes and the degradation of DNA into internucleosomal fragments in cells J. F. R. Kerr et al, Br. J. Cancer, 26:239 (1972); A. H. Wyllie, Nature, 284:555 (1980)!. Recent evidence has indicated that before the occurrence of morphologic changes and death itself in the spontaneous or induced apoptosis, the cells undergo substantial alterations in both phenotypic and functional properties. These include an activation of endogenous endonucleases, the expression of molecular markers, and a loss or increase in protein expression C. Zhang et al, J. Immunol., 157:3980-3987 (1996)!. Although molecular alterations are closely associated with apoptosis, their precise role in the process of apoptotic cell death remains to be understood.
In an effort to detect apoptotic cells, several different assays and assay reagents have been utilized. Apoptotic cells can be visualized by flow cytometry using fluorescent DNA stains I. Schmid et al, Cytometry, 15:12-20 (1994)!, DNA agarose gel electrophoresis A. H. Wyllie, cited above!, or by in situ nick translation R. R. Jonker et al., NATO ASI Series, H67:355-362 (1993)!. However, these assays and reagents have not proved adequate for distinguishing cells from early stages of apoptosis from those which are late apoptotic and necrotic. For example, the use of anti-tubulin antibody, which stains the intracellularly-located microtubules, has been described. O'Brien et al, Cytometry, 28:81-89 (1997)!. However, this antibody is not specific for apoptotic cells, but rather recognizes any cell having a disrupted membrane.
What is needed in the art is an efficient method for identifying and distinguishing early apoptotic and late apoptotic/necrotic cells.